Heparin is a highly sulfated free form glycosaminoglycan that exists in the intracellular granule of mast cells. Heparan sulfate is a less sulfated glycan part of proteoglycan molecules that are distributed on the cell surface and are important structural and functional components of the extracellular matrix of all mammalian cells. Heparin and heparan sulfate are linear, polydisperse, highly negative-charged polysaccharide chains composed of alternating uronate and hexasamine saccharides joined by (1xe2x86x924) glycosidic linkage. They have a molecule weight range from about 4000 to about 30000 Da.
Heparin has been widely employed as an anticoagulant and antithrombotic drug. The anticoagulant action of heparin resides in its interaction with antithrombin III via a specific pentasaccharide sequence which in turn accelerates the binding and inhibitory activity of antithrombin toward the serine proteases, thrombin, and Factor Xa in the coagulation cascade (Olson et al. 1991; Olson et al. 1992; Olson et al. 1994).
Heparin has other beneficial uses that are not related to its anticoagulant activity. Examples include treating inflammatory lesions and ischemia/reperfusion (I/R) injury syndromes in pulmonary and myocardial infarction, stroke, traumatic shock, thrombolytic therapy or solid organ transplantations and operations; treating airway allergenic bronchoconstriction or bronchial asthma; treating hemorrhagic, hypovolemic, septic shock and related syndromes; treating atherosclerosis and cancer metastasis; and treating viral infection and wound healing. The non-anticoagulant effects of heparin protects microvascular structures against degradation, preserves myocardial contractility, and the function of heart, lung, liver, gastrointestinal tract and kidney, reduces brain injury and improves immune function.
Heparin is isolated from porcine or bovine mucosa or bovine lung tissue for medicinal use. Heparin/heparan sulfate are very heterogeneous because of the complexity and nature of their biosynthetic pathway. Further, the composition of heparin/heparan sulfate varies significantly depending on the cellular source and stages of growth and development. Specifically, less than about 30% of the isolated heparin bears the specific pentasaccharide sequence necessary to interact with antithrombin. The rest of the heparin has essentially no anticoagulant activity. The active part of single heparin molecules of differing lengths is surrounded by large areas of less or different activity.
The use of such heterogeneous heparin as an anticoagulant or antithrombic drug has been linked to side effects such as hemorrhagic complications, thrombocytopenia, alopecia, osteoporosis, and adverse lipolysis. As many as half of patients receiving heparin for a period over 6 months develop clinically significant osteoporosis. Essentially all patients treated with heparin exhibit a transient thrombocytopenia, and approximately 5% of those patients persist in that state for the duration of therapy. These side effects of heparin significantly limit the clinical use of this important anticoagulant, particularly for long term use.
Many heparin derivatives aimed at overcoming the problems have been investigated. Low Molecule Weight Heparin (LMWH), obtained by depolymerization and fractionation of unfractionated crude heparin has a lower average molecular weight (4000-6000 Da) and is claimed to have improved properties over unfractionated heparin. These include higher antithrombotic/antihemostatic ratio, higher bioavailability from injection site, longer duration of effect, lower propensity to bind acute phase plasma proteins as well as macrophage and the vascular endothelium and many other tissue proteins, and reduced side effects (Lane D., 1989, London: Edward Arnold; Barrowcliffe et al. 1992). However, these claimed improved properties are still controversial because, although the molecular weight are within quite narrow range, the composition of LMWH is still as complicated as unfractionated heparin.
Likewise, the anticoagulant fraction of heparin can cause complications such as hemorrhage for the non-anticoagulant applications of heparin. This has motivated interest in selectively decoupling the anticoagulant activities of heparin from its many non-anticoagulant properties. Strategies have included N-desulfation/N-acetylation, O-desulfation, and carboxyl reduction by chemical modification (U.S. Pat. Nos. 6,127,347, 4,916,219, 5,090,910, 5,795,875). Usually some of the anticoagulant activity remains, albeit significantly reduced. This may still pose an adverse effect for hypersensitive patients. Also, in some cases the desired activity is lost along with the anticoagulant activity due to the fractionation.
It is therefore highly desirable to have an efficient method and apparatus for separating and isolating the anticoagulant and non-anticoagulant fractions of heparin. One alternative would be affinity-purification with antithrombin. However, the use of natural blood-derived antithrombin has been prohibitive because of safety issues and the cost of scale-up. Currently, there is no cost-effective alternative to blood-derived antithrombin. The ideal method and apparatus would be safe, inexpensive, readily available, simple to use and capable of providing heparin or heparan sulfate in yields large enough to be clinically significant.
A patient receiving cardiopulmonary surgery, cardiac catheterization and hemodialysis, and other types of operations and therapies that cause blood coagulation, is often administered heparin anticoagulant to prevent blood clotting. A second agent is then typically administered to neutralize or remove the heparin from blood at the conclusion of medical procedures to prevent overshoot and potentially harmful persistent lack of coagulation. This strategy both moderates the continued action of heparin and prevents the accompanying severe side effects. This is commonly achieved by administration of protamine, an arginine-rich basic polypeptide from salmon sperm, which does not exist in human cells and forms a strong complex with heparin. In fact, protamine is the only choice so far for heparin neutralization. However, protamine administration has a number of unwanted side-effects including a modest elevation of blood pressure, severe allergic response, hypotension, complement activation, leukopenia, thrombocytopenia, pulmonary edema and vasoconstriction, and anaphylactic shock. The incidence of mild reactions to the use of protamine is as high as 16%, and that of severe reactions is between 0.2% to 3% (Holland et al., 1984; Cook et al., 1992). Therefore there is intense need for a safe and effective substitution of protamine.
Heparin-binding platelet factor 4 (PF4) has been proposed as a scavenger for neutralizing or reversing the anticoagulant effect (U.S. Pat. No. 5,482,923). However, since it is a blood system protein with multiple functions, it impacts numerous other biological processes. These include inhibition of angiogenesis and endothelial cell proliferation, modulation of host immunoreactivity, and enhancement of thrombomodulin anticoagulant function and inflammatory reactions that are in addition to binding and neutralization of heparin and anticoagulation. These complicated functions represent a major hurdle to its clinical application. The administration of PF4 to patients has resulted in serious granulocytopenia in humans (U.S. Pat. No. 5,801,063).
Heparinase I, an enzyme that degrades highly sulfated heparin chains containing 1-4 linkage to 2-O-sulfated iduronic acid residues, has also been evaluated clinically to remove heparin. Heparinase I also degrades natural heparan sulfate on the cell surface and in the extracellular matrix, which has critical biological functions. Heparinase is a slow-acting enzyme from bacteria and must remain stable for efficacy of activity. It needs specific conditions for maximal stability and activity. Longer term, it causes an immune response. These features collectively have dampened enthusiasm for its wide use.
Lactoferrin, or its fragment, has been proposed as a potential reagent to neutralize heparin. Lactoferrin is a natural blood component from neutrophil secondary granules, and is also found in milk, tears and saliva. It is an iron-binding protein and exhibits a 55% homology with the serum iron transporting protein, transferrin. Its heparin binding ability has only partially been characterized. Lactoferrin elutes from immobilized heparin at lower salt concentrations than that of antithrombin, indicating it has a lower affinity and lacks specificity for binding anticoagulant heparin. In addition, its physiological roles in the regulation of host defense and inflammation also remain unclear. These are expected to limit its clinical utility for neutralizing heparin.
A device with immobilized antithrombin for removing heparin from whole blood during extracorporeal circulation has also been proposed, but the instability, the safe issue and the cost of preparation and production of antithrombin impaired its use. In summary, although numerous alternatives have been attempted for protamine, none have exhibited the cost-benefit ratio equal to it. Alternatives that are potent in reversing the heparin anticoagulation effect are also bioactive and impact many other physiological processes, are generally more expensive, and lack specificity for the antithrombin-binding, anticoagulant motif. A cheap, safe and more effective alternative would be of benefit.
One aspect of the present invention is a method for purifying heparin with anticoagulant and antithrombotic activity comprising contacting the affinity matrix with a mixture comprising anticoagulant heparin or heparan sulfate and separating the non-bound material from the bound material.
A further aspect of the invention is an affinity matrix for purifying anticoagulant heparin or heparan sulfate comprising a fibroblast growth factor immobilized on a support.
Another aspect of the invention is a method of making an affinity matrix for isolating anticoagulant heparin or heparan sulfate comprising providing a fibroblast growth factor and immobilizing it on a support.
Further included in the invention is a method of preparing FGF7 protein in bacteria, comprising transforming a bacterium with a recombinant vector encoding a GST-FGF7 fusion protein and culturing the bacteria in a media containing a salt.
The invention also provides a method of neutralizing anticoagulation catalyzed by heparin, a heparin mimic, or a heparin derivative, comprising contacting the heparin, the heparin mimic, or the heparin derivative with a fibroblast growth factor.
SEQ ID NO.1 is the nucleotide sequence coding for the Fibroblast Growth Factor 7 (FGF7) from rat (rattus norvegicus, Genbank access No. 022182).
SEQ ID NO.2 is the amino acid sequence corresponding to SEQ ID NO.1.
SEQ ID NO.3 is the nucleotide sequence coding for the fusion protein Glutathione-S-Transferase-Fibroblast Growth Factor 7 (GST-FGF7).
SEQ ID NO.4 is the amino acid sequence corresponding to SEQ ID NO.3.